Constitutive expression of murine c-FLIPR causes autoimmunity in aged mice.

Death receptor-mediated apoptosis is a key mechanism for the control of immune responses and dysregulation of this pathway may lead to autoimmunity. Cellular FLICE-inhibitory proteins (c-FLIPs) are known as inhibitors of death receptor-mediated apoptosis. The only short murine c-FLIP splice variant is c-FLIPRaji (c-FLIPR). To investigate the functional role of c-FLIPR in the immune system, we used the vavFLIPR mouse model constitutively expressing murine c-FLIPR in all hematopoietic compartments. Lymphocytes from these mice are protected against CD95-mediated apoptosis and activation-induced cell death. Young vavFLIPR mice display normal lymphocyte compartments, but the lymphocyte populations alter with age. We identified reduced levels of T cells and slightly higher levels of B cells in 1-year-old vavFLIPR mice compared with wild-type (WT) littermates. Moreover, both B and T cells from aged vavFLIPR animals show activated phenotypes. Sera from 1-year-old WT and transgenic animals were analysed for anti-nuclear antibodies. Notably, elevated titres of these autoantibodies were detected in vavFLIPR sera. Furthermore, tissue damage in kidneys and lungs from aged vavFLIPR animals was observed, indicating that vavFLIPR mice develop a systemic lupus erythematosus-like phenotype with age. Taken together, these data suggest that c-FLIPR is an important modulator of apoptosis and enforced expression leads to autoimmunity.

Phosphorylation of Atg5 by the Gadd45ß-MEKK4-p38 pathway inhibits autophagy.

Autophagy is a lysosomal degradation pathway important for cellular homeostasis, mammalian development, cancer and immunity. Many molecular components of autophagy have been identified, but little is known about regulatory mechanisms controlling their effector functions. Here, we show that, in contrast to other p38 MAP kinase activators, the growth arrest and DNA damage 45 beta (Gadd45ß)-MAPK/ERK kinase kinase 4 (MEKK4) pathway specifically directs p38 to autophagosomes. This process results in an accumulation of autophagosomes through p38-mediated inhibition of lysosome fusion. Conversely, autophagic flux is increased in p38-deficient fibroblasts and Gadd45ß-deficient cells. We further identified the underlying mechanism and demonstrate that phosphorylation of the autophagy regulator autophagy-related (Atg)5 at threonine 75 through p38 is responsible for inhibition of starvation-induced autophagy. Thus, we show for the first time that Atg5 activity is controlled by phosphorylation and, moreover, that the spatial regulation of p38 by Gadd45ß/MEKK4 negatively regulates the autophagic process.

The role of c-FLIP splice variants in urothelial tumours.

Deregulation of apoptosis is common in cancer and is often caused by overexpression of anti-apoptotic proteins in tumour cells. One important regulator of apoptosis is the cellular FLICE-inhibitory protein (c-FLIP), which is overexpressed, for example, in melanoma and Hodgkin's lymphoma cells. Here, we addressed the question whether deregulated c-FLIP expression in urothelial carcinoma impinges on the ability of death ligands to induce apoptosis. In particular, we investigated the role of the c-FLIP splice variants c-FLIP(long) (c-FLIP(L)) and c-FLIP(short) (c-FLIP(S)), which can have opposing functions. We observed diminished expression of the c-FLIP(L) isoform in urothelial carcinoma tissues as well as in established carcinoma cell lines compared with normal urothelial tissues and cells, whereas c-FLIP(S) was unchanged. Overexpression and RNA interference studies in urothelial cell lines nevertheless demonstrated that c-FLIP remained a crucial factor conferring resistance towards induction of apoptosis by death ligands CD95L and TRAIL. Isoform-specific RNA interference showed c-FLIP(L) to be of particular importance. Thus, urothelial carcinoma cells appear to fine-tune c-FLIP expression to a level sufficient for protection against activation of apoptosis by the extrinsic pathway. Therefore, targeting c-FLIP, and especially the c-FLIP(L) isoform, may facilitate apoptosis-based therapies of bladder cancer in otherwise resistant tumours.

Hyaluronan oligosaccharide treatment of chondrocytes stimulates expression of both HAS-2 and MMP-3, but by different signaling pathways.

OBJECTIVE: Small hyaluronan (HA) oligosaccharides displace HA from the cell surface and induce cell signaling events. In articular chondrocytes this cell signaling is mediated by the HA receptor CD44 and includes stimulation of genes involved in matrix degradation such as matrix metalloproteinases (MMPs) as well as matrix repair genes including collagen type II, aggrecan and HA synthase-2 (HAS-2). The objective of this study was to determine whether stimulation of HAS-2 and MMP-3 by HA oligosaccharides is due to the activation of a single, cascading pathway or multiple signaling pathways. METHOD: Bovine articular chondrocytes were pre-treated with a variety of inhibitors of major signaling pathways prior to the addition of HA oligosaccharides. Changes in HA were monitored by real time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of HAS-2 mRNA, HA ELISA and HA accumulation at the cell surface. A 1900 base pair sequence containing the proximal promoter of HAS-2 was inserted into a luciferase reporter construct, transfected into human immortalized chondrocytes and assayed in a similar fashion. RESULTS: While our previous studies demonstrated that HA oligosaccharides stimulate MMP-13 activity via activation of p38 MAP kinase and NF-kappaB, inhibitors of these pathways did not affect the stimulation of HAS-2 mRNA expression. However, inhibiting the phosphatidylinositol-3-kinase pathway blocked HA oligosaccharide-mediated stimulation of HAS-2 yet had no effect on MMP-3. Wortmannin and LY294002 also blocked HA oligosaccharide-induced serine and threonine Akt phosphorylation. Treatment of transfected immortalized chondrocytes with HA oligosaccharides resulted in stimulation of HAS-2 mRNA, activation of Akt and enhanced luciferase activity-activity that was blocked by inhibitors of Akt phosphorylation. CONCLUSIONS: Changes in chondrocyte-matrix interactions by HA oligosaccharides induce altered matrix metabolism by the activation of least two distinct signaling pathways.

Different forms of cell death induced by putative BCL2 inhibitors.

Several inhibitors of BCL2 proteins have been identified that induce apoptosis in a variety of tumor cells, indicating their potential in cancer therapy. We investigated the specificity of six putative BCL2 inhibitors (obatoclax, gossypol, apogossypol, EM20-25, chelerythrine and ABT-737). Using cells deficient either for Bax/Bak or caspase-9, we found that only ABT-737 specifically targeted BCL2 proteins and induced apoptosis by activation of caspase-9, as only ABT-737 induced apoptosis was completely inhibited in cells deficient for Bax/Bak or caspase-9. Our data show that only ABT-737 is a specific BCL2 inhibitor and all other compounds investigated were not specific for BCL2 proteins. Furthermore, investigations of the effects of these compounds in primary chronic lymphocytic leukemic cells showed that all compounds induced certain biochemical hallmarks of apoptosis, such as release of cytochrome c and caspase cleavage. However, they all caused strikingly different ultrastructural changes. ABT-737 induced all the characteristic ultrastructural changes of apoptosis together with early rupture of the outer mitochondrial membrane, whereas obatoclax, chlelerythrine and gossypol induced pronounced mitochondrial swelling with formation of phospholipid inclusions. Therefore, we conclude that biochemical measurements used earlier to define apoptosis like mitochondrial release of cytochrome c and caspase cleavage, are insufficient to distinguish between classic apoptosis and other forms of cell death.

Impaired CD95-mediated apoptosis in autoimmunity and occurrence of a p22 Caspase-8 cleavage product in JIA.

BACKGROUND: Altered apoptosis can lead to autoimmune diseases such as systemic lupus erythematosus (SLE). Juvenile idiopathic arthritis (JIA) is another autoimmune disease characterized by autoinflammation. During this process activated T-cells accumulate in the synovial fluid. We hypothesized that resistance to CD95-mediated apoptosis could contribute to autoimmune phenotypes. PATIENTS/METHOD: We isolated highly activated T cells (CD45RO+ and CD95+) by magnetic separation from healthy controls, JIA patients and patients with other autoimmune diseases. In these purified cells, apoptosis was induced by stimulation with recombinant human soluble CD95 ligand (rhsCD95L) or dexamethasone and analyzed by flow cytometry. In addition, cleavage and expression of apoptosis mediators (Caspase-8 and -3) and regulators (FLIP, Bcl-2, Bcl-xL) were analyzed in mononuclear cells using immunoblot technique. RESULTS: Apoptosis upon CD95 stimulation, but not dexamethasone treatment, was reduced in JIA patients and patients with other autoimmune diseases compared to healthy controls. Additionally we observed a non-canonical cleavage pattern of Caspase-8 resulting in a p22 fragment and high expression of FLIP in SFMCs of patients with JIA. CONCLUSION: Expression and cleavage of proteins of the CD95 pathway is altered in JIA providing a possible explanation for resistance against death receptor-mediated apoptosis. Dexamethasone-induced apoptosis, however, is intact arguing against a general defect in apoptosis. The implications of the p22 fragment regarding apoptosis have to be further analyzed. The strong expression of FLIPShort in SFMCs may as well result from the highly activated status of the cells or be a feature of autoimmunity.

Mutational analyses of c-FLIPR, the only murine short FLIP isoform, reveal requirements for DISC recruitment.

Cellular FLICE-inhibitory protein (c-FLIP) proteins are known as potent inhibitors of death receptor-mediated apoptosis by interfering with caspase-8 activation at the death-inducing signaling complex (DISC). Among the three human isoforms, c-FLIP(long), c-FLIP(short) and c-FLIP(R), the latter isoform is poorly characterized. We report here the characterization of murine c-FLIP(R) and show that it is the only short c-FLIP isoform expressed in mice. By generating several mutants, we demonstrate that both death effector domains (DEDs) are required for DISC binding and the antiapoptotic function of c-FLIP(R). Surprisingly, the C-terminal tail is important for both protein stability and DISC recruitment. Three-dimensional modeling of c-FLIP(R) revealed a substantial similarity of the overall structures and potential interaction motifs with the viral FLIP MC159. We found, however, that c-FLIP(R) uses different structural motifs for its DISC recruitment. Whereas MC159 interferes with interaction and self-oligomerization of the DISC component FADD by its extensive hydrophilic surface, a narrow hydrophobic patch of c-FLIP(R) on the surface of DED2 is crucial for DISC association. Thus, despite the presence of similar tandem DEDs, viral and cellular FLIPs inhibit apoptosis by remarkably divergent mechanisms.

Analysis of biodegradation of copolymer dermis substitutes in the dorsal skinfold chamber of balb/c mice.

PEGT/PBT-block-copolymer dermis substitutes were inserted into dorsal skinfold chambers of balb/c mice (n=36). Scaffolding matrices with 3 different pore diameters (pore diameter: <75 micro m, 75-212 micro m and 250-300 micro m) were analyzed on days 7, 14, and 21 post implantation by scanning electron and light microscopy. The quantification of matrix fragmentation was performed using image-analytical software analySIS(R). The fragmentation rate in scaffolding matrices with a pore size of < 75 micro m was observed to be higher than in matrices of larger pore sizes. Image-analytical evaluation over 21 days revealed a reduction of the copolymer matrix by approximately 32% for the <75 micro m matrices, 23% for the 75-212 micro m matrices and 18% for the matrices, where pore size ranged between 250 micro m and 300 micro m. Twenty-one days after implantation, the matrix pores of 75-212 micro m and 250-300 micro m scaffolds were totally filled by vascularized fibrous tissue. Contrarily, an increased formation of foreign-body giant cells was observed in matrices with pore size <75 micro m. The pore size of the scaffolding PEGT/PBT dermis substitutes affects their degradative behaviour in vivo.

CD95 ligand mediates T-cell receptor-induced apoptosis of a CD4+ CD8+ double positive thymic lymphoma.

Tumors in the thymus can be of different cellular origin. Among the most common tumors are thymoma and lymphoma, which are derived from transformed thymic epithelial cells and transformed lymphocytes, respectively. Thymic lymphoma and their response to apoptotic stimuli are poorly characterized. Here, we analyse apoptosis events in the thymic lymphoma cell line Thy278, which expresses cell surface antigens characteristic of immature double positive thymocytes. Upon T-cell receptor (TCR)/CD3 stimulation, Thy278 cells die by apoptosis, similar as primary thymocytes during negative selection. Caspases are crucial for deletion of both Thy278 cells and normal thymocytes. Moreover, we show that deletion of primary thymocytes and Thy278 cells upon CD3 stimulation is considerably impaired by neutralizing CD95L antibody. Thus, our results not only demonstrate that TCR-induced apoptosis is still functional in transformed thymocytes, but also suggest that Thy278 cells are a helpful model for the molecular analysis of negative selection.

The role of CAP3 in CD95 signaling: new insights into the mechanism of procaspase-8 activation.

Formation of the CD95 (APO-1/Fas) death inducing signaling complex (DISC) plays a central role in CD95 signaling. Previously, CD95 DISC composition was analyzed by two-dimensional gel electrophoresis and four major cytotoxicity-associated proteins (CAP1-4) were found. CAP1 and CAP2 were defined to be unmodified and phosphorylated FADD, respectively. CAP4 was identified as procaspase-8a. CAP3, however, has remained elusive. In this study, we demonstrate that CAP3 is an intermediate of procaspase-8 processing. CAP3 is generated within seconds of DISC formation and subsequently processed to the prodomain of procaspase-8a that is known as p26 (CAP5). These findings lead to new insights into the mechanism of procaspase-8 processing and apoptosis initiation.

A comparative study of clinically well-characterized human atherosclerotic plaques with histological, chemical, and ultrastructural methods.

Atherosclerotic plaques (six cases) with well-documented clinical history were analysed using histology, scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive X-ray (EDX) spectroscopy, infrared spectroscopy (IR), thermogravimetry (TG), and high-resolution synchrotron X-ray diffraction. All samples contained about 60-70 wt% biological carbonated apatite (in dry state) in a nanocrystalline form with particle sizes of about 20 nm. Structurally, there are strong similarities to bone mineral. Ultrastructural investigations documented typical calcospherites, mineralisation processes starting at collagen fibrils and ring-shaped crystalline mineralised structures. There were no significant ultrastructural or chemical differences between the calcifications of individual patients.

[A rare case of a neuroendocrine carcinoma of the esophagus. Intermediate between a well-differentiated neuroendocrine carcinoma and a low-differentiated neuroendocrine carcinoma]. / Ungewöhnliches neuroendokrines Karzinom des Osophagus. Ubergangsform zwischen atypischem Karzinoid und niedrig differenziertem neuroendokrinen Karzinom.

We report a rare case of a neuroendocrine carcinoma located in the esophagus of a 62-year-old male patient. The initial diagnosis of a "small-cell tumor" was based on biopsy. Our diagnosis was based on the histomorphological examination of the resected material. Diagnostic criteria were the characteristic solid or clustered growth patterns, monomorphic cell nuclei, lack of necrosis, immunohistochemical detection of neuroendocrine markers like chromogranin, synaptophysin and neuron-specific enolase (NSE) as well as detection of cytoplasmic neuroendocrine granules by electron microscopy. In addition, we found an increased prolific activity by staining with Ki67 antigen. 30% of the cell nuclei displayed a positive reaction. Focal invasion of blood vessels was also detected. With 17 different chromosomal imbalances, comparative genomic hybridization (CGH) revealed a malignant tumor stage that was not visible at the microscopic level. According to the new WHO classification of neuroendocrine tumors the described tumor was identified as an intermediate between a well-differentiated neuroendocrine carcinoma and a low-differentiated neuroendocrine carcinoma.

[Elemental analysis of tattoo dyes-is there a potential risk from tattoo dyes?]. / Elementanalytische Untersuchungen von Tätowierungsfarbstoffen--Besteht eine potentielle Gefährdung durch Tätowierungsfarbstoffe?

BACKGROUND: Tattoo dyes in current use can cause foreign body reactions. METHODS: There are no rules regulating the composition of tattoo dyes. We performed elemental analysis on a series of dyes obtained from tattoo studios to determine if any dangerous materials were present. The composition of the dyes was determined using scanning electron microscopy in combination with energy dispersive X-ray microanalysis (EDS). This technique demonstrates bets the presence of elements of the sodium family and proves the presence of various metals. It provides no insight in chemical structures or bonds. RESULTS: Elemental analysis revealed multiple metallic components in the dyes; these materials may be responsible for persistent foreign body reactions even years after being placed in the skin. Silicon, aluminium, titanium and copper were found in various yellow, green and red dyes. The composition of various dyes of the same colour from different sources was highly variable. CONCLUSIONS: The tattoo dyes currently in use contain a number of components which cannot be regarded as "tissue inert". Chronic foreign body reactions can be expected even after many years.

[Aneurysms in arterio-venous synthetic shunts after dialysis therapy. Morphology and pathogenesis]. / Aneurysmen in arteriovenösen Kunststoffshunts nach Dialysetherapie. Morphologie und Pathogenese.

Synthetic grafts are used as arterio-venous fistulae (shunts) for dialysis therapy of patients with chronic renal insufficiency. Today grafts made of PTFE (polytetrafluorethylene) are commonly used. Grafts with diameter of 6 mm are implanted subcutaneously. In rare cases stenosis or thrombosis cause explantation of the grafts. Our results, obtained on 10 samples taken from surgery or autopsy, revealed that the main causes for graft explantation are destructions and perforations of the graft's wall caused by needle puncturing during dialysis. Single defects of the implants wall ranging from 200-500 micro m, and in some cases up to 1,5 mm are "repaired" by connective tissue. But greater defects cannot be compensated and are insufficiently covered by a network of loose connective tissue. These zones are under the predominated sites for development of false prosthetic aneurysm, which show parallel to posttraumatic aneurysms of vessels. Prosthetic aneurysms up to diameters of 3,2 cm were seen. Suitable choice of puncturing sites and puncturing technique can help to avoid destruction of the synthetic material.

[Ossification in lung metastases of primary colorectal adenocarcinomas]. / Ossifikation in Lungenmetastasen primärer kolorektaler Adenokarzinome.

Ossification of lung tissue is a rare phenomenon, which can be found in association with carcinoid tumors of the lung or pulmonary blastomas. Very rarely, ossifications are observed in lung metastases of extrathoracal epithelial tumors. In these cases, the most probable primary focus is a colorectal adenocarcinoma. Our question was, whether ossifications in lung metastases are pathognomonic for colorectal adenocarcinomas and how they can be pathogenetically arranged. A total of 15 lung metastases with ossifications from 5 patients suffering from a colorectal adenocarcinoma were examined by means of immunohistochemistry. Thereby, we found an increased expression of bone morphogenetic protein (BMP) 2/4 and osteonectin in tumor cells, as well as an increased stromatogenous expression of collagen type III. We conclude that there is strong evidence of a primary colorectal adenocarcinoma when ossifications in lung metastases are found. In these cases, a common metastatic spread of tumor cells and tumor stroma seems to be probable.

Urge incontinence and voiding postponement in children: somatic and psychosocial factors.

AIM: To analyse the number of urinary tract infections, uroflowmetry, behavioural symptoms and intrafamilial interaction in two groups of daytime wetting children in a paediatric and a child psychiatric unit. METHODS: Ninety-four children with either voiding postponement (52) or urge incontinence (42) were examined prospectively for history of urinary tract infections (UTIs), uroflowmetry, the syndrome scales of the Child Behaviour Checklist (CBCL 4/18-Achenbach) and the Family Adaptability and Cohesion Evaluation Scales (FACES-III) (Olson) questionnaire. RESULTS: Children with urge incontinence had a significantly higher rate of previous urinary tract infections (50%) than children with voiding postponement (19.2%; p < 0.001), who showed a high rate of plateau (12.2%) and staccato (20.4%) curves and were characterized by a wide variety of behavioural symptoms, including withdrawn (11.6%), aggressive (11.8%), delinquent (19.6%) behaviour and attention problems (13.7%). Clinically relevant behavioural scores were 4-10 times higher for the voiding postponers, and 2-3 times higher for children with urge incontinence. Furthermore, families of voiding postponers had significantly fewer balanced types of intrafamilial function (FACES-III). Problematic "rigid/disengaged" and "rigid/separated" types predominated. CONCLUSION: Urge incontinence is characterized by a higher rate of UTIs, a lower urine volume in uroflowmetry, a lower rate of behavioural scores in the clinical range and well-functioning families. Voiding postponement children, on the other hand, have a higher, though not significant, rate of abnormal uroflow curves, a wide variety of clinically relevant behavioural symptoms, which were significantly higher for attention and delinquent problems. Conduct problems predominated; only 13.7% of the children had attention problems in the clinical range. The findings lend empirical support to the entity of voiding postponement as an acquired or behavioural syndrome characterized by wetting in association with a delay of micturition and other externalizing conduct problems.

[Fatal cardiomyopathy in adult in polyglucosan body disease]. / Letale Kardiomyopathie bei adulter Polyglukosankörperkrankheit.

Adult polyglucosan body disease (APBD) is a rare genetic disorder, inherited in an autosomal recessive mode. The disease is caused by mutations of the gene coding for the glycogen-branching enzyme, which is essential for branching of polyglucose chains in the normal glycogen molecule. The age of clinical manifestation of the disease mostly is between 40 and 60 years and its course is slowly progressive. Characteristic globular deposits (polyglucosan bodies, PGB) can be detected in biopsies of skin and skeletal muscle as well as in the peripheral and central nervous system. Biochemically, PGBs consist of poorly branched glycogen molecules with abnormally long polysaccharide chains. We report the case of a 50-year-old female patient with APBD who suffered from neurological symptoms such as spastic tetraparesis, urinary incontinence, hypesthesia and dementia. She died unexpectedly of cardiac failure. At autopsy a severe cardiomyopathy with abundant PGBs in the heart muscle fibres could be proven as the cause of death. This observation shows that in addition to the known deposition of PGBs in nervous system and skeletal muscle, an involvement of the heart has to be considered in APBD as well.

Black spots of the parietal pleura: morphology and formal pathogenesis.

BACKGROUND: Dark incorporations (black spots) have been described in various organs. 'Black spots' seen as flat-to-nodular lesions of the parietal pleura are common findings in former miners. They represent areas of coal dust accumulation. An increased incorporation of asbestos fibres has been described in these areas, causing them to be seen as potential starting points for malignant mesotheliomas. OBJECTIVES: The aim of our examinations was to describe the morphology of black spots in order to understand their formal pathogenesis and discuss their role in the development of malignant mesotheliomas. MATERIALS: We report the results of the morphological and energy dispersive X-ray analysis of 12 black spots (4 surgical and 8 autopsy specimens) located in the parietal pleura. RESULTS: Black spots of the pleura develop in close correlation to lymphatic channels and blood vessels. Their formal pathogenesis is characterized by a mild fibrosis and an inflammatory reaction to the incorporated foreign particles. The proliferation of connective tissue may result in the formation of hyaline granulomas. Aluminum, silicone and sometimes fibres are also found in such areas. Mesothelial cells may be irritated. CONCLUSION: Although there are hints for an increased proliferation of mesothelial cells in some areas with black spots, our findings do not support the classification of black spots as an obligate early lesion in the development of malignant mesotheliomas.

A reporter-cell assay for the detection of BMP-2 immobilized on porous and nonporous materials.

Human recombinant bone morphogenetic protein-2 (rhBMP-2) immobilized on the surface of metal implants can facilitate osseointegration. Here, we describe a cell reporter assay useful for quantifying small amounts of immobilized rhBMP-2 on various materials. The peptide was dotted and heat-fixed on titanium, 316L stainless steel, nitrocellulose, or glass, and its distribution was monitored by in situ biotinylation followed by detection with the avidin-biotin method. Bioactivity of rhBMP-2 was demonstrated by means of a confluent layer of osteoblastic MC3T3-E1 cells that evenly covered rhBMP-2-free and rhBMP-2-loaded surface areas, as shown with epifluorescence microscopy of calcein acetoxymethyl (AM)-loaded cells. Expression of osteocalcin, fibronectin, actin, and vimentin increased where cells were located on rhBMP-2 dotted areas, but the signal:noise ratio was too low to bioassay the peptide. However, local pronounced expression of alkaline phosphatase was used to quantify BMP-2 in the range of 5-80 ng/dot by means of a cytochemical color reaction for alkaline phosphatase and image analysis of resulting dots. The lower detection limit was in the order nitrocellulose > glass > titanium > 316L steel. We conclude that the cell reporter assay is useful to assess biological activity of rhBMP-2 even after immobilization on three-dimensional implant materials.